A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Abstract Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been stymied by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections. Author summary Klebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae . These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae .